Vaccine compositions

ABSTRACT

The invention provides a DNA construct comprising a DNA sequence encoding a fusion protein of the formula: TetC-(Z) a -Het, wherein: TetC is the C fragment of tetanus toxin, or a protein comprising the epitopes thereof; Het is a heterologous protein, Z is an amino acid, and a is zero or a positive integer, provided that (Z) a  does not include the sequence Gly-Pro. The invention also provides replicable expression vectors containing the constructs, bacteria transformed with the constructs, the fusion proteins per se and vaccine compositions formed from the fusion proteins or attenuated bacteria expressing the fusion proteins.

This invention relates to DNA constructs, replicable expression vectorscontaining the constructs, bacteria containing the constructs andvaccines containing the bacteria or fusion proteins expressed therefrom.More particularly, the invention relates to novel DNA constructsencoding the C-fragment of tetanus toxin, and to fusion proteinscontaining tetanus toxin C-fragment.

It is known to prepare DNA constructs encoding two or more heterologousproteins with a view to expressing the proteins in a suitable host as asingle fusion protein. However, it has often been found that fusing twoproteins together in this way leads to an incorrectly folded chimaericprotein which no longer retains the properties of the individualcomponents. For example, the B-subunits of the Vibrio cholerae (CT-B)and E. coli (LT-B) enterotoxins are powerful mucosal immunogens butgenetic fusions to these subunits can alter the structure and propertiesof the carriers and hence their immunogenicity (see M. Sandkvist et al.J. Bacteriol. 169, pp4570-6, 1987, Clements et al. 1990 and M. Lipscombeet al. Mol. Microbiol. 5, pp 1385, 1990). Moreover, many heterologousproteins expressed in bacteria are not produced in soluble properlyfolded or active forms and tend to accumulate as insoluble aggregates(see C. Schein et al. Bio/Technology 6, pp 291-4, 1988 and R. Halenbecket al. Bio/Technology 7, pp 710-5, 1989.

In our earlier unpublished international patent applicationPCT/GB93/01617, it is disclosed that by providing a DNA sequenceencoding tetanus toxin C-fragment (TetC) linked via a “hinge region” toa second sequence encoding an antigen, the expression of the sequence inbacterial cells is enhanced relative to constructs wherein theC-fragment is absent. For example, the expression level of the fulllength P28 glutathione S-tranferase protein of S. mansoni when expressedas a fusion to TetC from the nirB promoter was greater than when the P28protein was expressed alone from the nirB promoter. The TetC fusion tothe full length P28 protein of S. mansoni was soluble and expressed inboth E. coli and S. typhimurium. In addition, the TetC-P28 fusionprotein was capable of being affinity purified by a glutathione agarosematrix, suggesting that the P28 had folded correctly to adopt aconformation still capable of binding to its natural substrate. It waspreviously considered that a hinge region, which typically is a sequenceencoding a high proportion of proline and/or glycine amino acids, isessential for promoting the independent folding of both the TetC and theantigenic protein fused thereto. However, it has now been discovered,surprisingly in view of the previous studies on CT-B and LT-B referredto above, that when the hinge region is omitted between the TetC and asecond antigen such as P28, the proteins making up the fusion do exhibitcorrect folding as evidenced by affinity purification on a glutathioneagarose matrix.

Accordingly, in a first aspect, the invention provides a DNA constructcomprising a DNA sequence encoding a fusion protein of the formulaTetC-(Z)_(a)-Het, wherein TetC is the C fragment of tetanus toxin, or aprotein comprising the epitopes thereof; Het is a heterologous protein;Z is an amino acid, and a is zero or a positive integer, provided that(Z)_(a) does not include the sequence Gly-Pro.

Typically (Z)_(a) is a chain of 0 to 15 amino acids, for example 0 to10, preferably less than 6 and more preferably less than 4 amino acids.

In one embodiment (Z)_(a) is a chain of two or three amino acids, theDNA sequence for which defines a restriction endonuclease cleavage site.

In another embodiment, a is zero.

Usually the group (Z)_(a) will not contain, simultaneously, both glycineand proline, and generally will not contain either glycine or proline atall.

In a further embodiment, (Z)_(a) is a chain of amino acids provided thatwhen a is 6 or more, (Z)_(a) does not contain glycine or proline.

The group (Z)_(a) may be a chain of amino acids substantially devoid ofbiological activity.

In a second aspect the invention provides a replicable expressionvector, for example suitable for use in bacteria, containing a DNAconstruct as hereinbefore defined.

In another aspect, the invention provides a host (e.g. a bacterium)containing a DNA construct as hereinbefore defined, the DNA constructbeing present in the host either in the form of a replicable expressionvector such as a plasmid, or being present as part of the hostchromosome, or both.

In a further aspect, the invention provides a fusion protein of the formTetC-(Z)_(a)-Het as hereinbefore defined, preferably in substantiallypure form, said fusion protein being expressible by a replicableexpression vector as hereinbefore defined.

In a further aspect the invention provides a process for the preparationof a bacterium (preferably an attenuated bacterium) which processcomprises transforming a bacterium (e.g. an attenuated bacterium) with aDNA construct as hereinbefore defined.

The invention also provides a vaccine composition comprising anattenuated bacterium, or a fusion protein, as hereinbefore defined, anda pharmaceutically acceptable carrier.

The heterologous protein “Het” may for example be a heterologousantigenic sequence, e.g. an antigenic sequence derived from a virus,bacterium, fungus, yeast or parasite.

Examples of viral antigenic sequences are sequences derived from a typeof human immunodeficiency virus (HIV) such as HIV-1 or HIV-2, the CD4receptor binding site from HIV, for example from HIV-1 or -2., hepatitisA, B or C virus, human rhinovirus such as type 2 or type 14, Herpessimplex virus, poliovirus type 2 or 3, foot-and-mouth disease virus(FMDV), rabies virus, rotavirus, influenza virus, coxsackie virus, humanpapilloma virus (HPV), for example the type 16 papilloma virus, the E7protein thereof, and fragments containing the E7 protein or itsepitopes; and simian immunodeficiency virus (SIV).

Examples of antigens derived from bacteria are those derived fromBordetella pertussis (e.g. P69 protein and filamentous haemagglutinin(FHA) antigens), Vibrio cholerae, Bacillus anthracis, and E.coliantigens such as E.coli heat Labile toxin B subunit (LT-B), E.coli K88antigens, and enterotoxigenic E.coli antigens. Other examples ofantigens include the cell surface antigen CD4, Schistosoma mansoni P28glutathione S-transferase antigens (P28 antigens) and antigens offlukes, mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, andmalaria parasites, eg. parasites of the genus plasmodium or babesia, forexample Plasmodium falciparum, and peptides encoding immunogenicepitopes from the aforementioned antigens.

Particular antigens include the full length Schistosoma mansoni P28, andoligomers (e.g. 2, 4 and 8-mers) of the immunogenic P28 aa 115-131peptide (which contains both a B and T cell epitope), and humanpapilloma virus E7 protein, Herpes simplex antigens, foot and mouthdisease virus antigens and simian immunodeficiency virus antigens.

The DNA constructs of the present invention may contain a promoter whoseactivity is induced in response to a change in the surroundingenvironment. An example of such a promoter sequence is one which hasactivity which is induced by anaerobic conditions. A particular exampleof such a promoter sequence is the nirB promoter which has beendescribed, for example in International Patent ApplicationPCT/GB92/00387. The nirB promoter has been isolated from E.coli, whereit directs expression of an operon which includes the nitrite reductasegene nirB (Jayaraman et al, J. Mol. Biol. 196, 781-788, 1987), and nirD,nirC, cysG (Peakman et al, Eur. J. Biochem. 191, 315323, 1990). It isregulated both by nitrite and by changes in the oxygen tension of theenvironment, becoming active when deprived of oxygen, (Cole, Biochem,Biophys. Acta. 162, 356-368, 1968). Response to anaerobiosis is mediatedthrough the protein FNR, acting as a transcriptional activator, in amechanism common to many anaerobic respiratory genes. By deletion andmutational analysis the part of the promoter which responds solely toanaerobiosis has been isolated and by comparison with otheranaerobically regulated promoters a consensus FNR-binding site has beenidentified (Bell et al, Nucl, Acids. Res. 17, 3865-3874, 1989; Jayaramanet al, Nucl, Acids, Res. 17, 135-145, 1989). It has also been shown thatthe distance between the putative FNR-binding site and the −10 homologyregion is critical (Bell et al, Molec. Microbiol.4, 1753-1763, 1990). Itis therefore preferred to use only that part of the nirB promoter whichresponds solely to anaerobiosis. As used herein, references to the nirBpromoter refer to the promoter itself or a part or derivative thereofwhich is capable of promoting expression of a coding sequence underanaerobic conditions. The preferred sequence, and which contains thenirB promoter is:

AATTCAGGTAAATTTGATGTACATCAAATGGTACCCCTTGCTGAATCGTTAAGG TAGGCGGTAGGGCC(SEQ ID NO: 1)

In a most preferred aspect, the present invention provides a DNAmolecule comprising the nirB promoter operably linked to a DNA sequenceencoding a fusion protein as hereinbefore defined.

In another preferred aspect of the invention, there is provided areplicable expression vector, suitable for use in bacteria, containingthe nirB promoter sequence operably linked to a DNA sequence encoding afusion protein as hereinbefore defined.

The DNA molecule or construct may be integrated into the bacterialchromosome, e.g. by methods known per se, and thus in a further aspect,the invention provides a bacterium having in its chromosome, a DNAsequence or construct as hereinbefore defined.

Stable expression of the fusion protein can be obtained in vivo. Thefusion protein can be expressed in an attenuated bacterium which canthus be used as a vaccine.

The attenuated bacterium may be selected from the genera Salmonella,Bordetella, Vibrio, Haemophilus, Neisseria and Yersinia. Alternatively,the attenuated bacterium may be an attenuated strain of enterotoxigenicEscherichia coli. In particular the following species can be mentioned:S.typhi—the cause of human typhoid; S.typhimurium—the cause ofsalmonellosis in several animal species; S.enteritidis—a cause of foodpoisoning in humans; S.choleraesuis—a cause of salmonellosis in pigs;Bordetella pertussis—the cause of whooping cough; HaemoDhilusinfluenzae—a cause of meningitis; Neisseria gonorrhoea the cause ofgonorrhoea; and Yersinia—a cause of food poisoning.

Examples of attenuated bacteria are disclosed in, for exampleEP-A-0322237 and EP-A-0400958, the disclosures in which are incorporatedby reference herein.

An attenuated bacterium containing a DNA construct according to theinvention, either present in the bacterial chromosome, or in plasmidform, or both, can be used as a vaccine. Fusion proteins (preferably insubstantially pure form) expressed by the bacteria can also be used inthe preparation of vaccines. For example, a purified TetC-P28 fusionprotein in which the TetC protein is linked via its C-terminus to theP28 protein with no intervening hinge region has been found to beimmunogenic on its own. In a further aspect therefore, the inventionprovides a vaccine composition comprising a pharmaceutically acceptablecarrier or diluent and, as active ingredient, an attenuated bacterium orfusion protein as hereinbefore defined.

The vaccine may comprise one or more suitable adjuvants.

The vaccine is advantageously presented in a lyophilised form, forexample in a capsular form, for oral administration to a patient. Suchcapsules may be provided with an enteric coating comprising, forexample, Eudragit “S”, Eudragit “L”, Cellulose acetate, Celluloseacetate phthalate or Hydroxypropylmethyl Cellulose. These capsules maybe used as such, or alternatively, the lyophilised material may bereconstituted prior to administration, e.g. as a suspension.Reconstitution is advantageously effected in buffer at a suitable pH toensure the viability of the organisms. In order to protect theattenuated bacteria and the vaccine from gastric acidity, a sodiumbicarbonate preparation is advantageously administered before eachadministration of the vaccine. Alternatively, the vaccine may beprepared for parenteral administration, intranasal administration orintramammary administration.

The attenuated bacterium containing the DNA construct or fusion proteinof the invention may be used in the prophylactic treatment of a host,particularly a human host but also possibly an animal host. An infectioncaused by a microorganism, especially a pathogen, may therefore beprevented by administering an effective dose of an attenuated bacteriumaccording to the invention. The bacterium then expresses the fusionprotein which is capable of raising antibody to the micro-organism. Thedosage employed will be dependent on various factors including the sizeand weight of the host, the type of vaccine formulated and the nature ofthe fusion protein.

An attenuated bacterium according to the present invention may beprepared by transforming an attenuated bacterium with a DNA construct ashereinbefore defined. Any suitable transformation technique may beemployed, such as electroporation. In this way, an attenuated bacteriumcapable of expressing a protein or proteins heterologous to thebacterium may be obtained. A culture of the attenuated bacterium may begrown under aerobic conditions. A sufficient amount of the bacterium isthus prepared for formulation as a vaccine, with minimal expression ofthe fusion protein occurring.

The DNA construct may be a replicable expression vector comprising thenirB promoter operably linked to a DNA sequence encoding the fusionprotein. The nirB promoter may be inserted in an expression vector,which already incorporates a gene encoding one of the heterologousproteins (e.g. the tetanus toxin C fragment), in place of the existingpromoter controlling expression of the protein. The gene encoding theother heterologous protein (e.g. an antigenic sequence) may then beinserted. The expression vector should, of course, be compatible withthe attenuated bacterium into which the vector is to be inserted.

The expression vector is provided with appropriate transcriptional andtranslational control elements including, besides the nirB promoter, atranscriptional termination site and translational start and stopcodons. An appropriate ribosome binding site is provided. The vectortypically comprises an origin of replication and, if desired, aselectable marker gene such as an antibiotic resistance gene. The vectormay be a plasmid.

The invention will now be illustrated but not limited, by reference tothe following examples and the accompanying drawings, in which:

FIG. 1 is a schematic illustration of the construction of plasmidpTECH1;

FIG. 2 illustrates schematically the preparation of the plasmidpTECH1-28 from the starting materials pTECH1 and PUC19-P28;

FIG. 3 illustrates schematically the preparation of the plasmidpTECH3-P28 from the starting materials plasmids pTECH1-P28 andpTETnir15;

FIGS. 4 and 5 are western blots obtained from bacterial cells harbouringthe pTECH3-P28 construct; and

FIG. 6 illustrates the glutathione affinity purification of TetC fusionsas determined by SDS-PAGE and Coomassie Blue Staining.

In accordance with the invention a vector was constructed to allowgenetic fusions to the C-terminus of the highly immunogenic C fragmentof tetanus toxin, without the use of a heterologous hinge domain. Afusion was constructed, with the gene encoding the protective 28 kDaglutathione S-transferase from Schistosoma mansoni. The recombinantvector was transformed into Salmonella typhimurium (SL338; rm⁺). Theresulting chimeric protein was stably expressed in a soluble form insalmonella as assessed by western blotting with fragment C andglutathione S-transferase antisera. Furthermore it was found that theP28 component of the fusion retains the capacity to bind glutathione.

The construction of the vector and the properties of the fusion proteinexpressed therefrom are described in more detail below.

EXAMPLE 1 Preparation of pTECH1

The preparation of pTECH1, a plasmid incorporating the nirB promoter andTetC gene, and a DNA sequence encoding a hinge region and containingrestriction endonuclease sites to allow insertion of a gene coding for asecond or guest protein, is illustrated in FIG. 1. Expression plasmidpTETnir15, the starting material shown in FIG. 1, was constructed frompTETtac115 (Makoff et al, Nucl. Acids Res. 17 10191-10202, 1989); byreplacing the EcoRI-ApaI region (1354bp) containing the lacI gene andtac promoter with the following pair of oligos 1 and 2:

Oligo-1 5′AATTCAGGTAAATTTGATGTACATCAAATGGTACCCCTTGCTGAATCGTTAAGGTAGGCGGTAGGGCC-3′ (SEQ ID NO: 2)

Oligo-2 3′-GTCCATTTAAACTACATGTAGTTTACCATGGGGAACGACTTAGCAATTCCATCCGCCATC-5′ (SEQ ID NO: 3)

The oligonucleotides were synthesised on a Pharmacia Gene Assembler andthe resulting plasmids confirmed by sequencing (Makoff et al,Bio/Technology 7, 1043-1046, 1989).

The pTETnir15 plasmid was then used for construction of the pTECH1plasmid incorporating a polylinker region suitable as a site forinsertion of heterologous DNA to direct the expression of fragment Cfusion proteins. pTETnir15 is a known pAT153-based plasmid which directsthe expression of fragment C. However, there are no naturally occurringconvenient restriction sites present at the 3′-end of the TetC gene.Therefore, target sites, preceded by a hinge region, were introduced atthe 3′-end of the TetC coding region by means of primers SEQ ID NO: 4and SEQ ID NO: 5 tailored with “add-on” adapter sequences (Table 1),using the polymerase chain reaction (PCR) [K. Mullis et al, Cold SpringHarbor Sym. Quant. Biol. 51, 263-273 1986]. Accordingly, pTETnir15 wasused as a template in a PCR reaction using primers corresponding toregions covering the SacII and BamHI sites. The anti-sense primer inthis amplification was tailored with a 38 base 5′-adaptor sequence. Theanti-sense primer was designed so that a sequence encoding novel XbaI,SpeI and BamHI sites were incorporated into the PCR product. Inaddition, DNA sequences encoding additional extra amino acids includingproline were incorporated (the hinge regions) and a translation stopcodon signal in frame with the fragment C open reading frame.

The PCR product was gel-purified and digested with SacII and BamHI, andcloned into the residual 2.8 kb vector pTETnir15 which had previouslybeen digested by SacII and BamHI. The resulting plasmid purified fromtransformed colonies and named pTECH 1 is shown in FIG. 1. Heterologoussequences such as the sequence encoding the Schistosoma mansoni P28glutathione S-transferase (P28) were cloned into the XbaI SpeI and BamHIsites in accordance with known methods.

The DNA sequence of the plasmid pTECH1 is shown in the sequence listingas SEQ ID NO: 6.

TABLE 1 DNA SEQUENCES OF OLIGONUCLEOTIDES UTILISED IN THE CONSTRUCTIONOF THE TETC-HINGE VECTORS A). Primer 1. Sense PCR (21mer). (SEQ ID NO:4)                   SacII      5′AAA GAC TCC GCG GGC GAA GTT-3′            TETANUS TOXIN C FRAGMENT SEQ. B). Primer 2. Anti-Sense PCRPrimer (64mer). (SEQ ID NO: 5)           BamHI  STOP  SpeI       XbaI5′-CTAT GGA TCC TTA ACT AGT GAT TCT AGA     HINGE REGION     GGG CCC CCGCCC GTC GTT GGT CCA ACC     TTC ATC GGT -3′       TETANUS TOXIN CFRAGMENT SEQ. 3′-END

EXAMPLE 2 Construction of pTECH1-P28

A P28 gene expression cassette was produced by PCR using pUC19-P28 DNA(a kind gift from Dr R Pierce, Pasteur Institute, Lille) as template.Oligonucleotide primers were designed to amplify the full length P28gene beginning with the start codon and terminating with the stop codon.In addition, the sense and antisense primers were tailored with therestriction sites for XbaI and BamHI respectively. The primers are shownin the sequence listing as SEQ ID NO: 7 and SEQ ID NO: 8.

The product was gel-purified and digested with XbaI and BamHI and thencloned into pTECH1 which had previously been digested with these enzymesand subsequently gel-purified. The DNA sequence of pTECH1-P28 is shownin sequence listing as SEQ ID NO: 9.

Expression of the TetC-Hinge-P28 Fusion Protein

Several bacterial strains, namely S. typhimirium strains SL 5338 (A.Brown et al, J.Infect.Dis. 155, 86-92, 1987) and SL3261 and E. coli(TG2) were transformed with pTECH1-P28 by means of electroporation.SL3261 strains harbouring the pTECH1-P28 plasmid have been deposited atthe National Collection of Type Cultures, 61 Colindale Avenue, London,NW9 5HT, UK under the accession number NCTC 12833. A strain of SL3261containing the pTECH1 plasmid has been deposited under accession numberNCTC 12831. The identity of recombinants was verified by restrictionmapping of the plasmid DNA harboured by the cells. Further expression ofthe TetC-P28 fusion protein was then evaluated by SDS-PAGE and westernblotting of bacterial cells harbouring the construct It was found thatthe fusion protein remains soluble, cross-reacts with antisera to bothTetC and P28, and is also of the expected molecular weight, 80kDal, fora full length fusion.

The fusion protein was stably expressed in E.coli (TG2) and S.typhimurium (SL5338, SL3261) as judged by SDS-PAGE and western blotting.Of interest was a band of 50kDal which co-migrates with the TetC-Hingeprotein alone and cross-reacts exclusively with the anti-TetC sera isvisible in a western blot. As the codon selection in the hinge regionhas been designed to be suboptimal, the rare codons may cause pausesduring translation which may occasionally lead to the prematuretermination of translation, thus accounting for this band.

Affinity Purification of the TetC-P28 Fusion

Glutathione is the natural substrate for P28, a glutathioneS-transferase. The amino acid residues involved in binding glutathioneare thought to be spatially separated in the primary structure of thepolypeptide and brought together to form a glutathione binding pocket inthe tertiary structure (P. Reinemer et al. EMBO, J8, 1997-2005, 1991).In order to gauge whether the P28 component of the fusion has foldedcorrectly to adopt a conformation capable of binding glutathione, itsability to be affinity purified on a glutathione-agarose matrix wastested. The results obtained (not shown) demonstrated that TetC-P28 canindeed bind to the matrix and the binding is reversible, as the fusioncan be competitively eluted with free glutathione.

EXAMPLE 3 Construction of TECH3-P28

The plasmid pTECH1-P28 directs the expression of the S. mansoni P28protein as a C-terminal fusion to fragment C from tetanus toxinseparated by a heterologous hinge domain. Expression of the fusionprotein is under the control of the nirB promoter. The vector pTECH3-P28was in part constructed from the plasmid pTETnir15 by the polymerasechain reaction (PCR) using the high fidelity thermostable DNA polymerasefrom Pyrococcus fusorius, which possesses an associated 3′5′ exonucleaseproofreading activity. The sequence of steps is summarised in FIG. 5. Inorder to generate a TetC-hingeless replacement cassette, the segment ofDNA from the unique SacII site within the TetC gene to the final codonwas amplified by means of the PCR reaction, using pTETnir15 as templateDNA. The primers used in the PCR amplification are shown in the sequencelisting as SEQ ID NO: 10 and SEQ ID NO: 11. The antisense primer in thisamplification reaction was tailored with an XbaI recognition sequence.

The amplification reaction was performed according to the manufacturer'sinstructions (Stratagene, La Jolla, Calif., USA). The product wasgel-purified, digested with SacII and XbaI, and then cloned into theresidual pTECH1-P28 vector which had been previously digested with therespective enzymes SacII and XbaI. The resulting vector was designatedpTECH3-P28. The DNA sequence of pTECH3-P28 is shown in the sequenclisting as SEQ ID NO: 12.

EXAMPLE 4 Transformation of S. typhimurium SL5338 (galE r⁻m⁺) withpTECH3-P28, and Analysis of the Transformants

S. typhimurium SL5338 (galE r⁻m⁺) were cultured in either L or YT brothand on L-agar with ampicillin (50 g/ml) if appropriate and weretransformed with the pTECH3-P28 plasmid. The transformation protocol wasbased on the method described by MacLachlan and Sanderson. (MacLachlanPR and Sanderson KE, 1985. Transformation of Salmonella typhimurium withplasmid DNA : differences between rough and smooth strains. J.Bacteriology 161, 442-445).

A 1ml overnight culture of S. typhimurium SL5338 (r⁻m³⁰; Brown A,Hormaeche CE, Demarco de Hormaeche R, Dougan G, Winther M, Maskell D,and Stocker BAD, 1987. J. Infect.Dis. 155, 86-92) was used to inoculate100 ml of LB broth and shaken at 37° C. until the culture reachedOD₆₅₀=0.2. The cells were harvested at 3000×g and resuspended in 0.5volumes if ice-cold 0.1M MgCl₂. The cells were pelleted again andresuspended in 0.5 volumes of ice-cold CaCl₂. This step was repeatedonce more and the cells resuspended in 1 ml of 0.1M CaCl₂ to which wasadded 50 μl of TES (50 mM Tris, 10 mM EDTA, 50 mM NaCl, pH 8.0). Thecells were incubated on ice for 45 to 90 minutes. To 150μl of cells wasadded 100ng of plasmid DNA in 1-2 μl. The mixture was incubated on icefor 30 minutes prior to heat-shock at 42° C. for 2 minutes, andimmediate reincubation on ice for 1 minute. To the transformed mixturewas added 2 ml of LB broth and incubated for 1.5 hours to allowexpression of the ampicillin drug resistance gene, B-lactamase.Following incubation 20 μl and 200 μl of cells were spread on to LB agarplates containing 50 μg/ml of ampicillin. The plates were dried andincubated at 37° C. overnight.

The identity of recombinants was verified by restriction mapping of theplasmid DNA and by western blotting with antisera directed against TetCand P28.

SDS-PAGE and Western Blotting

Expression of the TetC fusions was tested by SDS-PAGE and westernblotting. S. typhimurium SL5338 (galE r⁻m⁺) bacterial cells containingthe pTECH3-P28 plasmid and growing in mid-log phase, with antibioticselection, were harvested by centrifugation and the proteinsfractionated by 10% SDS-PAGE. The proteins were transferred to anitrocellulose membrane by electroblotting and reacted with either apolyclonal rabbit antiserum directed against TetC or the full length P28protein. The blots were then probed with goat anti-rabbit Ig conjugatedto horse-radish peroxidase (Dako, High Wycombe, Bucks, UK) and developedwith 4-chloro-1-napthol). The results of the western blottingexperiments are shown in FIGS. 4 and 5; FIG. 4 illustrating the resultsof probing with rabbit anti-TetC polyclonal antiserum and FIG. 5illustrates the results of probing with rabbit anti-P28 polyclonalantiserum. In each case lanes 1, 2 and 3 are independent clones ofSL5338 (pTECH3-P28), lanes 4, 5 and 6 are SL5338 (pTECH1-P28) and lane 7is SL5338 (pTETnir15). The molecular weight markers are indicated. Fromthe results, it is evident that the fusion protein remains soluble,reacts with antisera to both TetC and P28, and is also of the expectedmolecular weight, 80 kDal, for a full length fusion (FIG. 4).Furthermore the fusion protein appears to be stably expressed.

Glutathione-Agarose Affinity Purification

Glutathione is the natural substrate for P28, a glutathioneS-transferase. The amino acid residues involved in binding glutathioneare thought to be spatially separated in the primary structure of thepolypeptide and brought together to form a glutathione binding pocket inthe tertiary structure. In order to gauge whether the P28 component ofthe fusion has folded correctly to adopt a conformation capable ofbinding glutathione, we tested its ability to be affinity purified on aglutathione agarose matrix. Bacterial cells containing pTECH3-P28 andexpressing the TetC full length P28 gene fusion were grown to log phase,chilled on ice, and harvested by centrifugation at 2500×g for 15 min at4C. The cells were resuspended in {fraction (1/15)}th the originalvolume of ice-cold phosphate buffered saline (PBS) and lysed bysonication in a MSE Soniprep 150 (Gallenkamp, Leicester, UK). Theinsoluble material was removed by centrifugation and to the supernatantwas added ⅙ volume of a 50% slurry of pre-swollen glutathione-agarosebeads (Sigma, Poole, Dorset, UK). After mixing gently at roomtemperature for 1 hour the beads were collected by centrifugation at1000×g for 10 secs. The supernatant was discarded and the beadsresuspended in 20 volumes of cold PBS-0.5% Triton X100 and the beadscollected again by centrifugation. The washing step was repeated threemore times. The fusion protein was eluted by adding 1 volume of SDS-PAGEsample buffer. For comparison purposes, a similar procedure was followedwith bacterial cells containing the PTECH1-P28 plasmid from whichTetC-hinge-P28 fusion protein is expressed. Extracts from clonescontaining either plasmid were compared using SDS-PAGE and the resultsare shown in FIG. 6. In FIG. 6, lanes 1, 2 and 3 are clones of SL5338(pTECH1-P28) whereas lanes 4, 5 and 6 are independent clones of SL 5338(pTECH3-P28).

The results suggest that the TetC-P28 fusion protein can indeed bind tothe matrix and the binding is reversible regardless of the absence of aheterologous hinge domain (data not shown) It is possible that a peptidesequence present at the C-terminus of TetC may in fact impartflexibility to this particular region.

12 1 68 DNA Escherichia coli 1 aattcaggta aatttgatgt acatcaaatggtaccccttg ctgaatcgtt aaggtaggcg 60 gtagggcc 68 2 68 DNA ArtificialSequence misc_feature (1)..(68) Oligo-1 sequence 2 aattcaggta aatttgatgtacatcaaatg gtaccccttg ctgaatcgtt aaggtaggcg 60 gtagggcc 68 3 60 DNAArtificial Sequence misc_feature (1)..(60) Oligo-2 sequence 3 gtccatttaaactacatgta gtttaccatg gggaacgact tagcaattcc atccgccatc 60 4 21 DNAClostridium tetani 4 aaagactccg cgggcgaagt t 21 5 64 DNA Clostridiumtetani 5 ctatggatcc ttaactagtg attctagagg gccccggccc gtcgttggtccaaccttcat 60 cggt 64 6 3754 DNA Artificial Sequence misc_feature(1)..(3754) plasmid pTECH1 6 ttcaggtaaa tttgatgtac atcaaatggt accccttgctgaatcgttaa ggtaggcggt 60 agggcccaga tcttaatcat ccacaggaga ctttctgatgaaaaaccttg attgttgggt 120 cgacaacgaa gaagacatcg atgttatcct gaaaaagtctaccattctga acttggacat 180 caacaacgat attatctccg acatctctgg tttcaactcctctgttatca catatccaga 240 tgctcaattg gtgccgggca tcaacggcaa agctatccacctggttaaca acgaatcttc 300 tgaagttatc gtgcacaagg ccatggacat cgaatacaacgacatgttca acaacttcac 360 cgttagcttc tggctgcgcg ttccgaaagt ttctgcttcccacctggaac agtacggcac 420 taacgagtac tccatcatca gctctatgaa gaaacactccctgtccatcg gctctggttg 480 gtctgtttcc ctgaagggta acaacctgat ctggactctgaaagactccg cgggcgaagt 540 tcgtcagatc actttccgcg acctgccgga caagttcaacgcgtacctgg ctaacaaatg 600 ggttttcatc actatcacta acgatcgtct gtcttctgctaacctgtaca tcaacggcgt 660 tctgatgggc tccgctgaaa tcactggtct gggcgctatccgtgaggaca acaacatcac 720 tcttaagctg gaccgttgca acaacaacaa ccagtacgtatccatcgaca agttccgtat 780 cttctgcaaa gcactgaacc cgaaagagat cgaaaaactgtataccagct acctgtctat 840 caccttcctg cgtgacttct ggggtaaccc gctgcgttacgacaccgaat attacctgat 900 cccggtagct tctagctcta aagacgttca gctgaaaaacatcactgact acatgtacct 960 gaccaacgcg ccgtcctaca ctaacggtaa actgaacatctactaccgac gtctgtacaa 1020 cggcctgaaa ttcatcatca aacgctacac tccgaacaacgaaatcgatt ctttcgttaa 1080 atctggtgac ttcatcaaac tgtacgtttc ttacaacaacaacgaacaca tcgttggtta 1140 cccgaaagac ggtaacgctt tcaacaacct ggacagaattctgcgtgttg gttacaacgc 1200 tccgggtatc ccgctgtaca aaaaaatgga agctgttaaactgcgtgacc tgaaaaccta 1260 ctctgttcag ctgaaactgt acgacgacaa aaacgcttctctgggtctgg ttggtaccca 1320 caacggtcag atcggtaacg acccgaaccg tgacatcctgatcgcttcta actggtactt 1380 caaccacctg aaagacaaaa tcctgggttg cgactggtacttcgttccga ccgatgaagg 1440 ttggaccaac gacgggccgg ggccctctag aatcactagttaaggatccg ctagcccgcc 1500 taatgagcgg gctttttttt ctcgggcagc gttgggtcctggccacgggt gcgcatgatc 1560 gtgctcctgt cgttgaggac ccggctaggc tggcggggttgccttactgg ttagcagaat 1620 gaatcaccga tacgcgagcg aacgtgaagc gactgctgctgcaaaacgtc tgcgacctga 1680 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaagtctggaaac gcggaagtca 1740 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcggtcgttcggc tgcggcgagc 1800 ggtatcagct cactcaaagg cggtaatacg gttatccacagaatcagggg ataacgcagg 1860 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaaccgtaaaaagg ccgcgttgct 1920 ggcgtttttc cataggctcc gcccccctga cgagcatcacaaaaatcgac gctcaagtca 1980 gaggtggcga aacccgacag gactataaag ataccaggcgtttccccctg gaagctccct 2040 cgtgcgctct cctgttccga ccctgccgct taccggatacctgtccgcct ttctcccttc 2100 gggaagcgtg gcgctttctc aatgctcacg ctgtaggtatctcagttcgg tgtaggtcgt 2160 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcagcccgaccgct gcgccttatc 2220 cggtaactat cgtcttgagt ccaacccggt aagacacgacttatcgccac tggcagcagc 2280 cactggtaac aggattagca gagcgaggta tgtaggcggtgctacagagt tcttgaagtg 2340 gtggcctaac tacggctaca ctagaaggac agtatttggtatctgcgctc tgctgaagcc 2400 agttaccttc ggaaaaagag ttggtagctc ttgatccggcaaacaaacca ccgctggtag 2460 cggtggtttt tttgtttgca agcagcagat tacgcgcagaaaaaaaggat ctcaagaaga 2520 tcctttgatc ttttctacgg ggtctgacgc tcagtggaacgaaaactcac gttaagggat 2580 tttggtcatg agattatcaa aaaggatctt cacctagatccttttaaatt aaaaatgaag 2640 ttttaaatca atctaaagta tatatgagta aacttggtctgacagttacc aatgcttaat 2700 cagtgaggca cctatctcag cgatctgtct atttcgttcatccatagttg cctgactccc 2760 cgtcgtgtag ataactacga tacgggaggg cttaccatctggccccagtg ctgcaatgat 2820 accgcgagac ccacgctcac cggctccaga tttatcagcaataaaccagc cagccggaag 2880 ggccgagcgc agaagtggtc ctgcaacttt atccgcctccatccagtcta ttaattgttg 2940 ccgggaagct agagtaagta gttcgccagt taatagtttgcgcaacgttg ttgccattgc 3000 tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggcttcattcagct ccggttccca 3060 acgatcaagg cgagttacat gatcccccat gttgtgcaaaaaagcggtta gctccttcgg 3120 tcctccgatc gttgtcagaa gtaagttggc cgcagtgttatcactcatgg ttatggcagc 3180 actgcataat tctcttactg tcatgccatc cgtaagatgcttttctgtga ctggtgagta 3240 ctcaaccaag tcattctgag aatagtgtat gcggcgaccgagttgctctt gcccggcgtc 3300 aacacgggat aataccgcgc cacatagcag aactttaaaagtgctcatca ttggaaaacg 3360 ttcttcgggg cgaaaactct caaggatctt accgctgttgagatccagtt cgatgtaacc 3420 cactcgtgca cccaactgat cttcagcatc ttttactttcaccagcgttt ctgggtgagc 3480 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagggcgacacgga aatgttgaat 3540 actcatactc ttcctttttc aatattattg aagcatttatcagggttatt gtctcatgag 3600 cggatacata tttgaatgta tttagaaaaa taaacaaataggggttccgc gcacatttcc 3660 ccgaaaagtg ccacctgacg tctaagaaac cattattatcatgacattaa cctataaaaa 3720 taggcgtatc acgaggccct ttcgtcttca agaa 3754 730 DNA Schistosoma mansoni 7 tagtctagaa tggctggcga gcatatcaag 30 8 30DNA Schistosoma mansoni 8 ttaggatcct tagaagggag ttgcaggcct 30 9 4378 DNAArtificial Sequence misc_feature (1)..(4378) plasmid pTECH1-P28 9ttcaggtaaa tttgatgtac atcaaatggt accccttgct gaatcgttaa ggtaggcggt 60agggcccaga tcttaatcat ccacaggaga ctttctgatg aaaaaccttg attgttgggt 120cgacaacgaa gaagacatcg atgttatcct gaaaaagtct accattctga acttggacat 180caacaacgat attatctccg acatctctgg tttcaactcc tctgttatca catatccaga 240tgctcaattg gtgccgggca tcaacggcaa agctatccac ctggttaaca acgaatcttc 300tgaagttatc gtgcacaagg ccatggacat cgaatacaac gacatgttca acaacttcac 360cgttagcttc tggctgcgcg ttccgaaagt ttctgcttcc cacctggaac agtacggcac 420taacgagtac tccatcatca gctctatgaa gaaacactcc ctgtccatcg gctctggttg 480gtctgtttcc ctgaagggta acaacctgat ctggactctg aaagactccg cgggcgaagt 540tcgtcagatc actttccgcg acctgccgga caagttcaac gcgtacctgg ctaacaaatg 600ggttttcatc actatcacta acgatcgtct gtcttctgct aacctgtaca tcaacggcgt 660tctgatgggc tccgctgaaa tcactggtct gggcgctatc cgtgaggaca acaacatcac 720tcttaagctg gaccgttgca acaacaacaa ccagtacgta tccatcgaca agttccgtat 780cttctgcaaa gcactgaacc cgaaagagat cgaaaaactg tataccagct acctgtctat 840caccttcctg cgtgacttct ggggtaaccc gctgcgttac gacaccgaat attacctgat 900cccggtagct tctagctcta aagacgttca gctgaaaaac atcactgact acatgtacct 960gaccaacgcg ccgtcctaca ctaacggtaa actgaacatc tactaccgac gtctgtacaa 1020cggcctgaaa ttcatcatca aacgctacac tccgaacaac gaaatcgatt ctttcgttaa 1080atctggtgac ttcatcaaac tgtacgtttc ttacaacaac aacgaacaca tcgttggtta 1140cccgaaagac ggtaacgctt tcaacaacct ggacagaatt ctgcgtgttg gttacaacgc 1200tccgggtatc ccgctgtaca aaaaaatgga agctgttaaa ctgcgtgacc tgaaaaccta 1260ctctgttcag ctgaaactgt acgacgacaa aaacgcttct ctgggtctgg ttggtaccca 1320caacggtcag atcggtaacg acccgaaccg tgacatcctg atcgcttcta actggtactt 1380caaccacctg aaagacaaaa tcctgggttg cgactggtac ttcgttccga ccgatgaagg 1440ttggaccaac gacgggccgg ggccctctag aatggctggc gagcatatca aggttatcta 1500ttttgacgga cgcggacgtg ctgaatcgat tcggatgact cttgtggcag ctggtgtaga 1560ctacgaagat gagagaatta gtttccaaga ttggccaaaa atcaaaccaa ctattccaga 1620cggacgattg cctgcagtga aagtcactga tgatcatggg cacgtgaaat ggatgttaga 1680gagtttggct attgcacggt atatggcgaa gaaacatcat atgatgggtg aaacagacga 1740ggaatactat agtgttgaaa agttgattgg tcatgctgaa gatgtagaac atgaatatca 1800caaaactttg atgaagccac aagaagagaa agagaagata accaaagaga tattgaacgg 1860caaagttcca gttcttctca atatgatctg cgaatctctg aaagggtcga caggaaagct 1920ggctgttggg gacaaagtaa ctctagctga tttagtcctg attgctgtca ttgatcatgt 1980gactgatctg gataaaggat ttctaactgg caagtatcct gagatccata aacatcgaga 2040aaatctgtta gccagttcac cgcgtttggc gaaatattta tcgaacaggc ctgcaactcc 2100cttctaagga tccgctagcc cgcctaatga gcgggctttt ttttctcggg cagcgttggg 2160tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg 2220ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc 2280tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg 2340taaagtctgg aaacgcggaa gtcagcgctc ttccgcttcc tcgctcactg actcgctgcg 2400ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 2460cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 2520gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 2580tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 2640ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 2700atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag 2760gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 2820tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 2880cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 2940cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 3000tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 3060cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 3120cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 3180gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 3240gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 3300gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 3360ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 3420atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 3480agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 3540ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 3600tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat 3660ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 3720caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 3780gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 3840atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 3900accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata gcagaacttt 3960aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 4020gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 4080tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 4140aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 4200ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 4260aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat 4320tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcaagaa 4378 1021 DNA Clostridium tetani 10 aaagactccg cgggcgaagt t 21 11 30 DNAClostridium tetani 11 ttatctagag tcgttggtcc aaccttcatc 30 12 4366 DNAArtificial Sequence misc_feature (1)..(4366) plasmid pTECH3-P28 12ttcaggtaaa tttgatgtac atcaaatggt accccttgct gaatcgttaa ggtaggcggt 60agggcccaga tcttaatcat ccacaggaga ctttctgatg aaaaaccttg attgttgggt 120cgacaacgaa gaagacatcg atgttatcct gaaaaagtct accattctga acttggacat 180caacaacgat attatctccg acatctctgg tttcaactcc tctgttatca catatccaga 240tgctcaattg gtgccgggca tcaacggcaa agctatccac ctggttaaca acgaatcttc 300tgaagttatc gtgcacaagg ccatggacat cgaatacaac gacatgttca acaacttcac 360cgttagcttc tggctgcgcg ttccgaaagt ttctgcttcc cacctggaac agtacggcac 420taacgagtac tccatcatca gctctatgaa gaaacactcc ctgtccatcg gctctggttg 480gtctgtttcc ctgaagggta acaacctgat ctggactctg aaagactccg cgggcgaagt 540tcgtcagatc actttccgcg acctgccgga caagttcaac gcgtacctgg ctaacaaatg 600ggttttcatc actatcacta acgatcgtct gtcttctgct aacctgtaca tcaacggcgt 660tctgatgggc tccgctgaaa tcactggtct gggcgctatc cgtgaggaca acaacatcac 720tcttaagctg gaccgttgca acaacaacaa ccagtacgta tccatcgaca agttccgtat 780cttctgcaaa gcactgaacc cgaaagagat cgaaaaactg tataccagct acctgtctat 840caccttcctg cgtgacttct ggggtaaccc gctgcgttac gacaccgaat attacctgat 900cccggtagct tctagctcta aagacgttca gctgaaaaac atcactgact acatgtacct 960gaccaacgcg ccgtcctaca ctaacggtaa actgaacatc tactaccgac gtctgtacaa 1020cggcctgaaa ttcatcatca aacgctacac tccgaacaac gaaatcgatt ctttcgttaa 1080atctggtgac ttcatcaaac tgtacgtttc ttacaacaac aacgaacaca tcgttggtta 1140cccgaaagac ggtaacgctt tcaacaacct ggacagaatt ctgcgtgttg gttacaacgc 1200tccgggtatc ccgctgtaca aaaaaatgga agctgttaaa ctgcgtgacc tgaaaaccta 1260ctctgttcag ctgaaactgt acgacgacaa aaacgcttct ctgggtctgg ttggtaccca 1320caacggtcag atcggtaacg acccgaaccg tgacatcctg atcgcttcta actggtactt 1380caaccacctg aaagacaaaa tcctgggttg cgactggtac ttcgttccga ccgatgaagg 1440ttggaccaac gactctagaa tggctggcga gcatatcaag gttatctatt ttgacggacg 1500cggacgtgct gaatcgattc ggatgactct tgtggcagct ggtgtagact acgaagatga 1560gagaattagt ttccaagatt ggccaaaaat caaaccaact attccagacg gacgattgcc 1620tgcagtgaaa gtcactgatg atcatgggca cgtgaaatgg atgttagaga gtttggctat 1680tgcacggtat atggcgaaga aacatcatat gatgggtgaa acagacgagg aatactatag 1740tgttgaaaag ttgattggtc atgctgaaga tgtagaacat gaatatcaca aaactttgat 1800gaagccacaa gaagagaaag agaagataac caaagagata ttgaacggca aagttccagt 1860tcttctcaat atgatctgcg aatctctgaa agggtcgaca ggaaagctgg ctgttgggga 1920caaagtaact ctagctgatt tagtcctgat tgctgtcatt gatcatgtga ctgatctgga 1980taaaggattt ctaactggca agtatcctga gatccataaa catcgagaaa atctgttagc 2040cagttcaccg cgtttggcga aatatttatc gaacaggcct gcaactccct tctaaggatc 2100cgctagcccg cctaatgagc gggctttttt ttctcgggca gcgttgggtc ctggccacgg 2160gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact 2220ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg 2280tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa 2340acgcggaagt cagcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 2400gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 2460ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 2520ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 2580acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 2640tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 2700ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc 2760ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 2820ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 2880actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 2940gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 3000tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 3060caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 3120atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 3180acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 3240ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 3300ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 3360tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 3420tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 3480gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 3540tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 3600tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 3660ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 3720tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 3780ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 3840gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 3900ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa aagtgctcat 3960cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 4020ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 4080ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 4140gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 4200ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 4260gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt 4320aacctataaa aataggcgta tcacgaggcc ctttcgtctt caagaa 4366

what is claimed is:
 1. A DNA construct comprising a DNA sequenceencoding a fusion protein of the formula TetC-(Z)_(a)-Het, wherein TetCis the C fragment of tetanus toxin, Het is a heterologous protein, Z isan amino acid, and a is 0 or a positive integer less than 4, providedthat (Z)_(a) does not include the sequence Gly-Pro, wherein the aminoterminus of the fusion protein is the TetC.
 2. A DNA construct accordingto claim 1 wherein (Z)_(a) is a chain of two or three amino acids, theDNA sequence for which defines a restriction endonuclease cleavage site.3. A DNA construct according to claim 1 wherein a is zero.
 4. A DNAconstruct according to claim 1 in which (Z)_(a) is free from glycineand/or proline.
 5. A DNA construct according to claim 1 wherein theheterologous protein Het is an antigenic sequence obtained from a virus,bacterium, fungus, yeast or parasite.
 6. A DNA construct according toclaim 5 wherein the heterologous protein Het is the Schistosoma mansoniP28 glutathione S-transferase antigen.
 7. A replicable expression vectorcontaining a DNA construct as defined in claim
 1. 8. A replicableexpression vector according to claim 7 suitable for use in bacteria. 9.A host cell having integrated into the chromosomal DNA thereof a DNAconstruct as defined in claim
 1. 10. A host cell according to claim 9which is a bacterium.
 11. A fusion protein of the formulaTetC-(Z)_(a)-Het, wherein TetC is the C fragment of tetanus toxin, Hetis a heterologous protein, Z is an amino acid, and a is 0 or a positiveinteger less than 4, provided that (Z)_(a) does not include the sequenceGly-Pro, wherein the amino terminus of the fusion protein is the TetC.12. A process for the preparation of a bacterium which process comprisestransforming a bacterium with a DNA construct as defined in claim
 1. 13.A process according to claim 12 wherein the bacterium is an attenuatedbacterium.
 14. A vaccine composition comprising a fusion protein asdefined in claim 11 and a pharmaceutically acceptable carrier.
 15. Avaccine composition comprising an attenuated bacterium expressing afusion protein as defined in claim 2; and a pharmaceutically acceptablecarrier.
 16. A replicable expression vector containing a DNA constructas defined in claim
 2. 17. A replicable expression vector according toclaim 16 suitable for use in bacteria.
 18. A host cell having integratedinto the chromosomal DNA thereof a DNA construct as defined in claim 2.19. A host cell according to claim 18 which is a bacterium.
 20. Avaccine composition comprising an attenuated bacterium containing a DNAconstruct as defined in claim 2; and a pharmaceutically acceptablecarrier.
 21. A fusion protein of the formula TetC-(Z)_(a)-Het, whereinTetC is the C fragment of tetanus toxin, Het is a heterologous protein,(Z)_(a) is a chain of two or three amino acids, the DNA sequence forwhich defines a restriction endonuclease cleavage site, wherein theamino terminus of the fusion protein is the TetC.
 22. A vaccinecomposition containing a fusion protein as defined in claim 21 and apharmaceutically acceptable carrier.